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in u complexes, thus giving no evidence for K complexes.I3 In IV, the signs of the ai are those expected for odd-alternant radicals, even in substituted IV. 10 It follows from Figure 2 that CIDNP is useful in analyzing complex nmr spectra of aromatic molecules, because adjacent protons become oppositely polarized. Also, the absolute signs of the nmr coupling constants can be determined from the multiplet effect, which prevails at low magnetic fields.6 (13) Reference 1 , p 50. (14) Address correspondence to : IBM Research Laboratory, San Jose, Calif. 95114.
Joachim Bargonl4 IBM Thomas J. Watson Research Center Yorktown Heights, New York 10598 Receined May 4 , 1971
Singlet Oxygen Analogs in Biological Systems. Peroxidase-Catalyzed Oxygenation of l,3-Dienes
Sir: The oxygenation of organic compounds by singlet molecular oxygen is a subject of considerable current interest. Exactly analogous processes of oxygenation take place in biological systems in reactions catalyzed by dioxygenases. Recently, the coupled oxygenation of 1,3-dienes by soybean lipoxidase, an enzyme whose mode of action is analogous to the “ene” reaction of singlet oxygen, has been reported.’ Results on the direct oxygenation of 1,3-dienes by horseradish peroxidase are reported here. A dilute suspension of 1,3-diphenylisobenzofuran (or other substrates discussed herein) was prepared by the addition of 0.1 M pH 4.0 acetate buffer (300 ml) t o a solution of the compound (10 mg) in acetone (30 ml containing 5 pl of Tween 80). When the suspension was incubated with hydrogen peroxide (20 p1 of 10 m M solution) and horseradish peroxidase (10 mg), the yellow-green fluorescence of the isobenzofuran disappeared within minutes and o-dibenzoylbenzene was isolated in 65 yield.3 Apart from 1,3-diphenylisobenzofuran, other 1,3-dienoid systems were also oxidized, although at a much reduced rate. After 4 hr of incubation under the above conditions, 2,3,4,5tetraphenylfuran yielded 1,2-diben~oylstilbene~ (1 1 %; 60 % of starting material was recovered) and anthracene gave anthraquinone4 (26 %; 38 of starting material was recovered). Tetracyclone and 9,lO-diphenylanthracene were not 0xidized.j The oxygenation of the furans by peroxidase is directly analogous to the dyesensitized photooxygenation reaction^.^^^ Further confirmation of the similarity between these modes of oxygenations is furnished by an analysis of the origins of the oxygen atoms in the products of these reactions.
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When l80-enriched (7.9 f 0.3 %) 1,3-diphenylisobenzofuran was oxidized in dichloromethane solution to o-dibenzoylbenzene in methylene-blue-sensitized photooxygenation, the product had an l80enrichment of 7.9 k 0.3%. Thus, photooxygenation of 1,3diphenylisobenzofuran leads to the retention of the ethereal oxygen atom in the product. When the same sample of 180-enriched 1,3-diphenylisobenzofuran was oxidized by horseradish peroxidase and hydrogen peroxide, the la0enrichment in the product was 8.1 f 0.3%, while incubation in buffer made up from H2180(5.0% enrichment) led to no incorporation (0.2 0 . 3 x ) of l80into the product. These observations are consistent with the participation of the same mode of oxygenation in the enzyme-catalyzed and photooxygenation reactions. Although dye-sensitized photooxygenation of anthracene in aprotic solvents results in endoperoxide formation,8similarity between peroxidasecatalyzed and dye- sensitized oxygenations of anthracene can be demonstrated by methylene-blue-sensitized oxidation in methanol-benzene (119) which yields anthraquinone as the major product. Although the catalysis of oxygenation by horseradish peroxidase can be attributed t o the intermediacy of singlet molecular oxygen (failure of tetracyclone and 9,lO-diphenylanthracene to react can be attributed to the lesser reactivity of these compounds, especially when in competition with quenching by groups on the enzyme), there are alternative explanations of which the participation of an enzyme-bound metal peroxy complex, discussed in detail below, offers an interesting as well as likely possibility. The ability of a transition metal peroxy complex (diperoxychromium(V1) oxide etherate) to oxygenate 1,3-dienesg in a reaction different from that of singlet oxygenlo has been demonstrated. The existence of a peroxide-peroxidase complex containing the elements of an oxygen molecule bonded t o a ferrous ion (“peroxidase compound 111” which has also been considered to be identical with oxygenated ferroperoxidase) has been established. 1 2 , 1 3 The ease with which transition metal ions react with hydrogen peroxide to form peroxy complexes14 suggests that peroxidase compound I11 and possibly other peroxideperoxidase complexes have a peroxyheme structure of the general type 1. The oxygenation reactions cata-
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(1) H. W . 4 . Chan,J. Amer. Chem. SOC.,93,2357 (1971). (2) Type 11, purchased from Sigma Chemical Co.; no reaction was observed when boiled enzyme was used. (3) All products discussed were isolated by chromatography and shown to be identical with authentic material by melting point and ir and mass spectra. (4) These incubations were performed on a scale five times that used for 1,3-diphenylisobenzofuran and with the addition of further aliquots of hydrogen peroxide after 1 and 2 hr. (5) These compounds crystallized out of the emulsion in 0.5 hr, during which time no reaction was observed. (6) C. Dufraisse and S. Ecary, C.R . Acad. Sci., 223,735 (1946). (7) J. Martel, ibid., 244, 626 (1957).
Journal of the American Chemical Society
1 93:18
1
2
lyzed by horseradish peroxidase may therefore be attributed to the formation of such a peroxy complex. The results discussed here do not, however, rule out other mechanisms of oxidation; in particular, the possibility that a dihydroxylation is involved cannot be excluded. In this context, the role of an oxoiron species (8) C. Dufraisse and M. Gerard, ibid., 201,428 (1935). (9) H. W . 4 . Chan, Chem. Commun., 1550 (1970). (10) 9,lO-Diphenylanthracene is not oxidized by the chromium peroxy complex, but anthracene is oxygenated to anthraquinone; the oxygen atom of 1,3-diphenylisobenzofuranis also retained on conversion to o-dibenzoylbenzene. l L (11) H. W . 3 . Chan, unpublished observations. (12) I . Yamazaki and L. H. Piette, Biochim. Bi0phj.s. Acta, 77, 47 (1963). (13) H. S. Mason, Proc. Int. Symp. Enzyme Chem., 1957, 223 (1958). (14) J. A. Connor and E. A . V. Ebsworth, Adcan. Inorg. C h e m Radiochem., 6 , 279 (1964).
September 8, 1971
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as the active oxidant in mixed-function oxygenases has recently been suggested. l6 By analogy with transition metal complexes of ethylene and acetylene, l7 where there is considerable decrease in C-C bond order with concomitant increase in metal-carbon bonding, structures 1 and 2 can be regarded as different (and extreme) forms representing a T complex between oxygen and a metal atom. The metal in structure 1 will have a formal oxidation state of two in excess of that in structure 2. Thus, complexes of transition metals with molecular oxygen are often considered to have the former structure. I 8 , l 9 Catalysis by metalloenzymes that mimic singlet oxygen may therefore involve complexes of type 1 or 2 as the “enzyme-bound singlet-oxygen.” The oxygenation reactions of diperoxychromium(V1) oxide etherate show that the oxygen moiety in peroxy complexes of metals in their higher oxidation states can have considerable “singlet” character. The chemical activity of metaloxygen complexes containing oxygen bonded to metal atoms in their lower oxidation states is being examined. Acknowledgment. The author is grateful to the Gulbenkian Foundation for a Research Fellowship at Churchill College, Cambridge, England, and to the United Kingdom Agricultural Research Council for financial support. (15) I
